Enzymes responsible for clinical resistance to aminoglycoside antibiotics are being isolated from R-factor bacteria, purified towards homogeneity, and characterized with respect to their physical and catalytic properties. The primary method of approach is to apply affinity chromatography to accomplish reaction mechanisms and to tabulate substrate and inhibitory properties of various aminoglycoside antibiotics and derivatives on Gentamicin Acetyltransferase, Kanamycin Acetyltransferase IV, Genetamicin Adenylyltransferase II, and Neomycin-Kanamycin Phosphotransferase II. The overall objective is to provide a quantitative description of antibiotic resistance at the molecular level in order to facilitate a search for specific inhibitors of these enzymes, to be used in efforts to counteract clinical resistance of pathogenic organisms to antibiotic therapy, to improve therapy by conventional antibiotics, and to make possible a comparative analysis of these unusual enzymes to more common enzymes which catalyze homologous reactions. Parameters governing the identification and bacterial production of these enzymes will be examined with a view towards understanding the origin, induction, and expression of R-factor DNA, and ultimately the epidemiology of resistant pathogens.